EP2626420A2 - Polynucleotide zur Codierung von MHC class I HLA-B7-beschränkten HTERT-Epitopen, Analoga davon oder Polyepitope - Google Patents

Polynucleotide zur Codierung von MHC class I HLA-B7-beschränkten HTERT-Epitopen, Analoga davon oder Polyepitope Download PDF

Info

Publication number
EP2626420A2
EP2626420A2 EP13165854.4A EP13165854A EP2626420A2 EP 2626420 A2 EP2626420 A2 EP 2626420A2 EP 13165854 A EP13165854 A EP 13165854A EP 2626420 A2 EP2626420 A2 EP 2626420A2
Authority
EP
European Patent Office
Prior art keywords
htert
hla
restricted
cells
polynucleotide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP13165854.4A
Other languages
English (en)
French (fr)
Other versions
EP2626420B1 (de
EP2626420A3 (de
Inventor
Pierre Langlade-Demoyen
Fransisco Garcia Pons
Olivier Adotevi
Sylvain Cardinaud
Christine Neuveut
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Original Assignee
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut Pasteur de Lille, Institut National de la Sante et de la Recherche Medicale INSERM filed Critical Institut Pasteur de Lille
Priority to EP13165854.4A priority Critical patent/EP2626420B1/de
Publication of EP2626420A2 publication Critical patent/EP2626420A2/de
Publication of EP2626420A3 publication Critical patent/EP2626420A3/de
Application granted granted Critical
Publication of EP2626420B1 publication Critical patent/EP2626420B1/de
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the field of anticancer therapy, and to the identification of immunogenic peptides derived from the human telomerase reverse transcriptase (hTERT).
  • the present invention relates to polynucleotides encoding hTERT epitopes restricted to MHC class I molecule, analogues thereof and polyepitopes containing such epitopes and/or analogues.
  • vectors and cells comprising such polynucleotides.
  • the present invention also concerns compositions comprising hTERT polypeptides, corresponding polynucleotides, vectors and cells, for use in the treatment and/or prevention of cancer.
  • Telomerase is a ribonucleoprotein complex, consisting of a protein component, TERT, and an RNA component (TR) containing the template for the synthesis of the repeat unit (T 2 AG 3 ) added onto the ends of chromosomes, that stabilizes the chromosomes during replication and prevent end-to-end fusion. Maintenance of a constant telomere length prevents cells from aging, and confers immortality ( Hahn et al. Nat Med 1999;5:1164-70 ). High hTERT activity was found in more than 85% of human cancers, whereas most normal adult human tissues show no or little telomerase activity ( Counter et al. Blood 1995;85:2315-20 ).
  • telomere fragments of hTERT could serve as tumor specific antigen(s) and this has been confirmed in several reports ( Vonderheide et al. Immunity 1999;10:673-9 ).
  • Recent data from Phase I clinical trials demonstrate the feasibility of vaccine against hTERT in HLA-A2 + patients, opening the way for use of hTERT for therapeutic vaccination ( Vonderheide et al. Clin Cancer Res 2004;10:828-39 ; Parkhurst et al. Clin Cancer Res 2004;10:4688-98 ).
  • the immunogenic hTERT peptides identified to date are restricted to one MHC allele HLA-A2.1, with only two initial reports on two HLA-supertypes, HLA-A3 and HLA-A24, represented respectively in 44.2% and 40% of the population.
  • the present invention identifies new epitopes derived from hTERT, restricted to a particular HLA which is different from HLA-A3 and HLA-A24.
  • HLA-B7 supertype which is expressed in about 25% of the population, and particularly to the second allele the most expressed in the human HLA-B population: the HLA-*B0702 (allele present in 15-20% of individuals in human population).
  • the gene of the isoform-1 of the telomerase is 4015 base pairs (bp) long (NCBI Accession number AF015950) and encodes a protein of 1132 amino acids (NCBI Accession number AAC51672.1) ( Figure 1 ).
  • the invention concerns a polynucleotide encoding a human telomerase reverse transcriptase (hTERT) peptide.
  • the encoded peptides are 9 amino acids in length (nonamer) or 10 amino acids in length (decamer), and the polynucleotide has hence 27 or 30 nucleotides.
  • the encoded peptide is less than 15 amino acids and the polynucleotide has less than 45 nucleotides.
  • the invention also concerns a polynucleotide encoding hTERT peptides that are epitopes, restricted to MHC class I molecule, especially epitopes suitable to induce an immune response restricted to HLA-B7.
  • the nucleotide sequence of the polynucleotide of the invention is, in a particular embodiment, limited to the sequence encoding the hTERT peptide.
  • Such peptides can be chosen from the group consisting of MPRAPRCRA (p1; amino acid residues 1 to 9), APRCRAVRSL (p4; amino acid residues 4 to 13), APSFRQVSCL (p68; amino acid residues 68 to 77), RPAEEATSL (p277; amino acid residues 277 to 285), RPSFLLSSL (p342; amino acid residues 342 to 350), RPSLTGARRL (p351; amino acid residues 351 to 360), DPRRLVQLL (p444, amino acid residues 444 to 452), FVRACLRRL (p464, amino acid residues 464 to 472), AGRNMRRKL (p966, amino acid residues 966 to 974), LPGTTLTAL (p1107, amino acid residues 1107 to 1115) and LPSPKFTIL (p1123, amino acid residues 1123 to 1131).
  • MPRAPRCRA p1; amino acid residues 1 to 9
  • APRCRAVRSL p4;
  • the invention especially concerns a polynucleotide encoding a HLA-B7-restricted hTERT epitope, chosen from the group consisting of RPSLTGARRL (p351), APSFRQVSCL (p68), APRCRAVRSL (p4), DPRRLVQLL (p444), FVRACLRRL (p464), AGRNMRRKL (p966), LPGTTLTAL (p1107) and h LPSPKFTIL (p1123).
  • RPSLTGARRL p351
  • APSFRQVSCL p68
  • APRCRAVRSL p4
  • DPRRLVQLL DPRRLVQLL
  • FVRACLRRL AGRNMRRKL
  • LPGTTLTAL LPGTTLTAL
  • h LPSPKFTIL p1123
  • an “epitope” is an antigenic determinant, i.e. the peptide site recognized by cells of the immune system (immune cells) and especially the site necessary to elicit an immune response.
  • the term epitope encompasses both linear epitope for which the consecutive amino acids (especially, 9 or 10) are recognized by immune cells and, conformational epitope for which immune cells recognize amino acids to the extent they adopt a proper configuration or conformation. Consequently, in some epitopes, the conformation (three dimensional structure) is as important as the amino acid sequence (primary structure).
  • MHC class I-restricted refers to the capacity for a particular peptide or epitope to have an affinity for a MHC (major histocompatibility complex) molecule of class I.
  • HLA-B7-restricted refers to the capacity for a particular peptide or epitope to have an affinity for this type of HLA molecule.
  • MHC genes encode cell surface polymorphic molecules that do not bind only foreign peptides but also can bind overexpressed or not self peptides or mutated self peptides, to display them on the cell surface of cell enabling their recognition by appropriate immune cells, especially T-cells.
  • Said MHC molecules referred to as H-2 in mice and HLA (Human Leucocyte Antigen) in humans, are classified as either class I molecules (designated HLA-A, B, or C) or class II molecules (designated DP, DQ or DR).
  • MHC class I molecules specifically bind CD8 molecules expressed on cytotoxic T lymphocytes (also named TCD8 + ), whereas MHC class II molecules specifically bind CD4 molecules expressed on helper T lymphocytes (TCD4 + ).
  • MHC class I molecules bind peptides derived from proteolytically degraded proteins especially endogenously synthesized proteins, by a cell. Small peptides obtained accordingly are transported into the endoplasmic reticulum where they associate with nascent MHC class I molecules before being routed through the Golgi apparatus and displayed on the cell surface for recognition by cytotoxic T lymphocytes.
  • the above-identified peptides have been shown on the one hand to bind either with high or medium affinity to MHC class I molecule, and on the other hand to be efficiently transported as a MHC/epitope complex to the cell surface of cells.
  • the MHC class I molecule is an MHC allele of the HLA-B7 supertype family; the hTERT epitope is said HLA-B7 supertype-restricted.
  • Said family encompasses alleles B0702, B0703, B0704, B0705, B1508, B3501, B3502, B3503, B51, B5301, B5401, B5501, B5502, B5601, B5602, B6701 and B7801, family from which the HLA-B0702 is preferred (HLA-B0702-restricted hTERT epitope).
  • a MHC stabilization assay may be used to test the affinity of a peptide for a particular HLA class I molecule (relative avidity), such as the one described in Firat et al. (1999. Eur. J. of Immunol. 29: 3112-3121 ), incorporated herein by reference. Briefly, MHC class I molecule-transfected cells are incubated overnight at 2 x 10 5 cells/well in 96-well plates in serum free medium AIM-V (Invitrogen Corp., Gibco), supplemented with 100ng/ml of human ⁇ 2-microglobulin,
  • MHC class I-restricted hTERT epitopes can be classified as having high relative affinity for MHC class I molecule: MPRAPRCRA (p1), APRCRAVRSL (p4) and APSFRQVSCL (p68).
  • MPRAPRCRA p1
  • APRCRAVRSL p4
  • APSFRQVSCL p68
  • MHC class I-restricted hTERT epitopes can be classified as having a medium relative affinity for MHC class I molecule: RPAEEATSL (p277), RPSFLLSSL (p342) and RPSLTGARRL (p351).
  • the present invention also relates to a polynucleotide encoding a MHC class I-restricted epitope analogue, i.e., epitopes having at least one amino acid substitution compared to a class I-restricted hTERT epitope as described above, especially HLA-B7-restricted epitope analogue.
  • a MHC class I-restricted epitope analogue i.e., epitopes having at least one amino acid substitution compared to a class I-restricted hTERT epitope as described above, especially HLA-B7-restricted epitope analogue.
  • analogue as defined herein relates to a peptide whose sequence is derived from a hTERT peptide as described above by at least one amino acid substitution, either conservative, semi-conservative or non-conservative.
  • An analogue is opposed to an epitope by the fact that its nucleotide and/or amino acid sequence is not found in the reference hTERT gene or protein disclosed in Figure 1 which are considered within the present application as the molecules of reference to define the so-called wild type peptides or polynucleotides.
  • Such an analogue may result from the substitution in the corresponding nucleotide sequence of one or several nucleic base(s) in the codon encoding said amino acid.
  • a polynucleotide analogue differs from its wild type counterpart (polynucleotide encoding hTERT peptide with the reference sequence from which the peptide analogue is derived from) by at least one substitution, and preferably one, two or three substitutions in the codon(s) encoding the amino acid residue to be substituted.
  • the APRRLVQLL peptide (called p444*) is derived from the p444 peptide by the substitution of the first amino acid residue (D -> A).
  • a particular analogue of a HLA-B7-restricted analogue has the same length or is shorter than the epitope from which it derives.
  • the hTERT epitopes described above or the analogues always conserve in their primary structure a proline (P) in position 2, and/or one of the following amino acids in the last C-terminal position: A, L, I, M, V, F, W or Y. Therefore, the hTERT peptides, including the analogues, have the following consensus sequence: X-P-X 6-7 -[ALIMVFWY], wherein X refer to any amino acid, X 6-7 refers to the number of amino acids and [ALIMVFWY] refers to one of these amino acids.
  • the amino acid substitution or the corresponding codon substitution in the polynucleotide is not located in the second position (or second codon).
  • no substitution is carried out in the C-terminal position, even though the last C-terminal amino acid can be replaced by an equivalent amino acid, i.e. either A, L, I, M, V, F, W or Y.
  • the substitution is located in the first amino acid position, wherein any amino acid is replaced by an alanine (A).
  • the last C-terminal amino acid of a decamer is deleted to give a nonamer, provided that the resulting nonamer maintains or adopts the X-P-X 6-7 -[ALIMVFWY] consensus sequence.
  • an amino acid selecting among A, L, I, M, V, F, W and Y is added at the C-terminal end of a nonamer to give rise to decamer, provided that the resulting decamer maintains or adopts the X-P-X 6-7 -[ALIMVFWY] consensus sequence.
  • the tridimensional conformation of the peptide analogue must be the same or slightly modified with respect to the one of the wild type counterpart, to ensure a correct folding of the analogue and its correct binding to the MHC class I molecule.
  • the MHC stabilization assay described above can be used to check that such constraints are fulfilled.
  • the resulting analogue has at least the same characteristics as its wild type counterpart, in terms of affinity for a particular MHC class I molecule, especially HLA-B7 molecule, and has essentially the same capacity to be transported as an epitope/MHC complex on the cell surface and/or has essentially the same capacity to elicit an immunogenic response when tested in the same conditions.
  • the starting peptide is a hTERT epitope having a medium affinity
  • the resulting analogue has a higher affinity than its wild type counterpart.
  • the analogue has a higher immunogenicity than its wild type counterpart.
  • the p444* peptide analogue quoted above has an increased affinity for MHC class I molecule compared to the p444 peptide from which it is derived from.
  • the T lymphocytes do not recognize said analogue that is used to stimulate said lymphocytes only, via antigen presenting cells.
  • the analogue as described above keeps its immunogenic behaviour, and is able to elicit an immune response against cells overexpressing hTERT epitopes, i.e. that CTLs recognize the wild type epitope, even if stimulated with an epitope analogue.
  • the lymphocytes stimulated by an epitope analogue of the invention do not react against cells, which do not overexpressed hTERT epitopes. Therefore, stimulated lymphocytes do not react with cells overexpressing other epitopes (cross reaction) or with cells expressing hTERT epitope as basal level (healthy cells).
  • all the characteristics mentioned above are in a HLA-B7 environment, and preferably in a HLA-B0702 context.
  • a conventional cytotoxicity assay may be performed by using a standard 4-5h 51 Cr release assay to test the capacity of stimulated lymphocytes to react against target cells, such as in the Firat publication ( 1999. Eur. J. of Immunol. 29: 3112-3121 ) incorporated herein by reference. Briefly, cell suspension containing CTLs, are activated with peptide (hTERT peptide or analogue in the present case) plus self MHC Class I molecule in vivo. Target cells (expressing or not the corresponding HTERT epitope) previously incubated with 51 Cr, are then incubated with activated lymphocytes.
  • peptide hTERT peptide or analogue in the present case
  • target cells expressing or not the corresponding HTERT epitope
  • the recognition of target cells by activated CTL leads to the apoptosis of target cells and the release of 51 Cr, wherein said release is proportional to the number of target cells killed.
  • An incubation of target cells with a control peptide is used as a negative control to calculate the spontaneous release of 51 Cr.
  • Specific percentage of lysis is calculated by subtracting non-specific lysis observed with the control peptide to lysis obtained with the peptides to be tested. The higher the percentage, the more targets have been killed by the CTL.
  • Specific lysis is determined at several ratios of Effector (CTL) to target cells (E:T). The specific lysis is calculated as the ratio between [experimental release - spontaneous release] and [total release - spontaneous release].
  • the present invention also concerns a polynucleotide encoding a polyepitope.
  • a polyepitope is defined as a polypeptide having at least two epitopes, chosen among the MHC class I-restricted, especially HLA-B7-restricted hTERT epitopes (p1, p4, p68, p277, p342, p351, p444, p464, p966, p1107 and p1123) and/or MHC class I-restricted, especially HLA-B7-restricted, epitope analogues of the invention.
  • the polynucleotide of the invention comprises or consists of at least two polynucleotide units encoding said epitopes or analogues.
  • a polynucleotide unit is defined as the coding sequence for an epitope or analogue of the invention as disclosed herein.
  • the invention particularly concerns a polynucleotide encoding a polyepitope, comprising at least two epitopes chosen among (a) RPSLTGARRL (p351), (b) APSFRQVSCL (p68), (c) APRCRAVRSL (p4), (d) DPRRLVQLL (p444), (e) FVRACLRRL (p464), (f) AGRNMRRKL (p966), (g) LPGTTLTAL (p1107) and (h) LPSPKFTIL (p1123), or their analogues as defined above.
  • RPSLTGARRL p351
  • APSFRQVSCL p68
  • APRCRAVRSL p4
  • DPRRLVQLL DPRRLVQLL
  • FVRACLRRL p464
  • AGRNMRRKL AGRNMRRKL
  • LPGTTLTAL LPGTTLTAL
  • LPSPKFTIL p1123
  • Another polynucleotide, encoding a polyepitope comprises at least one polynucleotidic unit chosen in the group of either a polynucleotide encoding a HLA-B7-restricted hTERT epitope corresponding to (a) RPSLTGARRL (p351), (b) APSFRQVSCL (p68), (c) APRCRAVRSL (p4), (d) DPRRLVQLL (p444), (e) FVRACLRRL (p464), (f) AGRNMRRKL (p966), (g) LPGTTLTAL (p1107) and (h) LPSPKFTIL (p1123) and/or their analogues as defined above, and at least one polynucleotidic unit chosen in the group of the polynucleotides encoding the sequence MPRAPRCRA (p1), RPAEEATSL (p277) or RPSFLLSSL (p342), or their analogues
  • the number of MHC class I restricted hTERT epitopes and/or analogues in the prepared polyepitope is limited to 30.
  • the number of HLA-B7 restricted hTERT epitopes and/or analogues is limited to approximately 30, and is preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30.
  • the number of HLA-B0702 restricted hTERT epitopes and/or analogues is limited to approximately 10, and is preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the polynucleotide encoding a polyepitope has 30 or less polynucleotide units, especially 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 or any number within this range.
  • polynucleotide units (and accordingly the epitopes) of the polynucleotide are consecutive.
  • the size of the nucleic sequence encoding the consecutive hTERT epitopes or analogues is less than 3000 bp, and preferably less than 2000 bp, 1000 bp, 500 bp, 400 bp, 300 bp, 200 bp or 100 bp.
  • the polynucleotide encoding the multiple epitopes consists of a nucleic acid molecule encoding a truncated or mutated form of the hTERT protein.
  • the truncated or mutated form of the hTERT protein is deprived of its catalytic activity, i.e., is not capable to direct the synthesis of the repeat unit (T 2 AG 3 ) at the ends of chromosomes, participating in the maintenance of telomere length.
  • Such a hTERT protein deprived of its catalytic activity i.e., the retrotranscriptase activity, is said non-functional.
  • the nucleic acid molecule encoding a truncated or mutated form of the hTERT protein lacks the catalytic activity domain of hTERT.
  • the nucleic acid molecule encoding a truncated form of the hTERT protein encodes a protein consisting of at least the 500 last C-terminal amino acids.
  • the polynucleotide encodes a hTERT protein that is deleted for amino acids 867 to 869 (VDD sequence), corresponding to nucleotides 2654 to 2662 of Figure 1 (wild-type) and providing nucleotide 2657 to be contiguous to nucleotide 2658 in Figure 9 representing the deletion site, or alternatively for amino acids 864 to 872, corresponding to nucleotides 2645 to 2671 of Figure 1 (wild-type) and providing nucleotide 2648 to be contiguous to nucleotide 2649 in Figure 10 representing the deletion site.
  • VDD sequence amino acids 867 to 869
  • nucleotides 2654 to 2662 of Figure 1 (wild-type) and providing nucleotide 2657 to be contiguous to nucleotide 2658 in Figure 9 representing the deletion site
  • amino acids 864 to 872 corresponding to nucleotides 2645 to 2671 of Figure 1 (wild-type) and providing nucleotide 2648 to be contiguous to nu
  • the encoded hTERT protein has a deletion that comprises at least the amino acid residues 867 to 869 i.e., that the deletion is larger than the 3 amino acid residues (VDD sequence).
  • VDD sequence 3 amino acid residues
  • the invention concerns a polynucleotide comprising or consisting of the nucleotide sequence as set forth in Figure 9 or Figure 10 .
  • the polynucleotide units encoding the multiple epitopes of the invention can be arranged consecutively, i.e., the 3' end of the first polynucleotide unit is directly linked to the 5' end of the second polynucleotide unit (and so on), resulting in a polynucleotide encoding a peptidic sequence exclusively composed of consecutive epitopes.
  • Such a polynucleotide can encode a polyepitope comprising or consisting of the p1, p4, p68, p277, p342, p351, p444, p464, p966, p1107 and p1123 peptides.
  • the polynucleotide encodes the following peptidic sequence MPRAPRCRAAPRCRAVRSLAPSFRQVSCLRPAEEAT SLRPSFLLSSLRPSLTGARRL, comprising thus 6 MHC class I-restricted, particularly HLA-B7restricted, hTERT epitopes.
  • the multiple epitopes of the invention can alternatively be separated by a one-amino acid spacer or a peptide spacer, i.e., meaning that the different polynucleotide units are separated by one or several codon(s) encoding respectively one or several amino acid(s).
  • spacers improving the processing of multiple epitopes 4 amino acid-peptides composed of an arginine (R) in the C terminal position and hydrophilic residues (A, K, D and/or T) in other positions are preferred.
  • 4 amino acid-peptides having a positively charged residue or an acidic residue in the C terminal position may be used, dependently or independently of hydrophilic residues (A, K, D and/or T) in other positions.
  • said spacers are internal processing sequences such as endosomal or lysosomal processing sequences, enabling the better processing of the multiple epitopes and avoiding the processing of new peptides resulting from overlapping cutting.
  • Such a separation having recourse to a spacer can be used to separate all or, to the contrary, part of the polynucleotide units and accordingly, all or part of the epitopes.
  • the order in which the epitopes are arranged can be determined by the man skilled in the art, according to the following criteria: some orders may facilitate either the transcription and/or the translation of the polynucleotide, may facilitate the transport of the resulting expressed polyepitope in the endoplasmic reticulum (ER), especially if the tridimensional conformation impacts the properties, and may facilitate the processing of the polyepitope in several epitopes or analogues and avoid the processing of overlapping epitopes.
  • some orders may facilitate either the transcription and/or the translation of the polynucleotide, may facilitate the transport of the resulting expressed polyepitope in the endoplasmic reticulum (ER), especially if the tridimensional conformation impacts the properties, and may facilitate the processing of the polyepitope in several epitopes or analogues and avoid the processing of overlapping epitopes.
  • the polyepitope of the invention enables the elicitation of a CTL response against at least one, especially against several epitopes or analogues contained in the polyepitope, simultaneously, in a single animal or human.
  • the polynucleotide encoding the polyepitope of the invention further comprises a polynucleotide encoding a target signal, operably linked to the polynucleotidic unit encoding the most N-terminal epitope of the at least two epitopes.
  • "Operably linked" as used herein means that the target signal (upstream sequence) is linked to the N-terminal epitope (downstream sequence) in a way enabling the targeted signal to be operational, i.e., enabling to target the polyepitope to the correct cellular compartment or domain. Therefore, the link between the two sequences allows each sequence to play its own function in different locations and/or different stages.
  • said target signal is an endoplasmic reticulum signal sequence, and allows the polyepitope to be directed to the ER, for proper processing and association with the MHC class I molecule.
  • a codon encoding a methionine residue is added upstream of the sequence encoding the most N-terminal epitope, to enable the correct translation of the polynucleotide, if the translational process requires an initiation codon.
  • Such a codon is added, only when the most N-terminal epitope does not possess a methionine residue in its first position.
  • the present invention also relates to a polynucleotide, according to the definitions given above, comprising or consisting of at least two polynucleotide units selected from the group consisting of
  • each amino acid can be encoded by the codon from the hTERT nucleotide reference sequence, or by any codon encoding said amino acid.
  • the invention also relates to a polynucleotide comprising or consisting of any combination of at least two of these polynucleotide units or analogues thereof, selected from the above group, wherein said polynucleotide unit or analogue encodes a MHC class I-restricted, especially a HLA-B7-restricted, hTERT epitope.
  • a recombinant vector comprising or consisting of a polynucleotide of the invention, as defined above, is also one object of the present invention.
  • the recombinant vector can be a vector for eucaryotic or procaryotic expression, such as a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector and especially a retroviral vector, or any expression vector.
  • An expression vector as defined herein is chosen to enable the production of an epitope or analogue or polyepitope as defined above, either in vitro or in vivo.
  • the vector of the invention can further comprise transcription regulation regions (including promoter, enhancer, ribosome binding site (RBS), polyA signal), a termination signal, a prokaryotic or eukaryotic origin of replication and/or a selection gene.
  • transcription regulation regions including promoter, enhancer, ribosome binding site (RBS), polyA signal
  • a termination signal e.g., a prokaryotic or eukaryotic origin of replication and/or a selection gene.
  • the features of the promoter can be easily determined by the man skilled in the art in view of the expression needed, i.e., constitutive, transitory or inducible, strong or weak, tissue-specific and/or developmental stage-specific promoter. Therefore, tissue-specific promoters can be chosen depending on the organ in which a composition containing this vector is administered, for example injected, and depending on the intensity of expression required.
  • the promoter is the CMV promoter (human cytomegalovirus).
  • Said vector can also comprise sequence enabling condition
  • the expression vectors of the invention may be viral vectors, and particularly viral expression vector, such as retroviral-derived, especially lentiviral-derived vectors such as HIV-, FIV- or SIV-derived vectors. More particularly, the lentiviral-derived vector is a human lentiviral-derived vector such as an HIV expression vector, particularly HIV-1 or HIV-2-derived vector.
  • a retroviral-derived vector comprises a retroviral vector genome, usually included in a DNA construct, such as a plasmid, and expressed in viral particles, wherein said retroviral vector genome comprises the elements necessary for the retrotranscription, particularly the LTRs possibly mutated including deleted in part, especially deleted in the U3 region.
  • the retroviral-derived vector contains all the nucleotide sequences encoding the full-length retroviral proteins. Possibly, it contains part of one or several of said nucleotide sequences providing it does not encode said proteins or functional fragments thereof.
  • Said DNA construct comprising said retroviral vector genome further comprises a DNA of interest recombined with the retroviral nucleotide sequences, said DNA of interest comprising or consisting of a polynucleotide of the invention.
  • the retroviral-derived vector genome comprises a DNA flap as described below and at least one polynucleotide of the invention.
  • the retroviral-derived vector is a HIV expression vector comprising a DNA flap as described below and at least one polynucleotide of the invention. HIV vectors express therefore only the nucleic acid(s) of interest, including the polynucleotides of the invention, contained between the two HIV LTRs and can thus accommodate large sequences up to 5-6 kb.
  • a particular embodiment of the invention is a HIV expression vector, and most particularly a HIV-1 or HIV-2 expression vector, wherein a HIV-1 LTR or respectively the HIV-2 LTR is deleted for the promoter and the enhancer of the U3 domain ( ⁇ U3).
  • This particular deletion has been previously shown to increase the expression of the nucleic acid(s) contained in the vector, and particularly when associated with a promoter.
  • the vector comprises a LTR deleted in the promoter and the enhancer of the U3 domain, a promoter such as a CMV or EF1 ⁇ promoter and a polynucleotide of the invention.
  • polynucleotide of the invention introduced in the retroviral-derived vector is included in an expression cassette.
  • a DNA flap (or Triplex as disclosed in WO 99/55892 , WO 01/27300 and WO 01/27304 ) is a nucleotide sequence of retroviral or retroviral-like origin comprising two essential regions, i.e., the cPPT (central polypurine tract) and the CTS (cis-acting termination region) regions, wherein the cPPT and CTS regions induce a three-stranded DNA structure.
  • a DNA flap suitable for the invention may be obtained from a retrovirus or retrovirus-like organism such as retrotransposon, prepared synthetically (chemical synthesis) or by amplification of the DNA flap from any retrovirus nucleic acid such as Polymerase Chain Reaction (PCR).
  • the retrovirus from which the DNA flap may be obtained, is particularly a retrovirus or a lentivirus, especially a human retrovirus or lentivirus and is in particular a HIV retrovirus, the CAEV (Caprine Arthritis Encephalitis Virus) virus, the EIAV (Equine Infectious Anaemia Virus) virus, the VISNA virus, the SIV (Simian Immunodeficiency Virus) virus or the FIV (Feline Immunodeficiency Virus) virus.
  • the DNA flap is obtained from an HIV retrovirus, for example HIV-1 or HIV-2 virus or any different isolate of these two types.
  • DNA flap is used isolated from its natural (viral genome) nucleotide context i.e., isolated from the pol gene in which it is naturally contained in a retrovirus. Therefore, the DNA flap is used, in the present invention, deleted from the unnecessary 5' and 3' parts of the pol gene and is recombined with sequences of different origin.
  • the DNA flap acts as a cis-determinant of the vector nuclear import, and is of great interest for the recombination and the integration of nucleic acid(s) into both non-dividing and dividing cells.
  • Expression retroviral-derived vector, especially HIV derived-vectors, including the DNA flap sequence (TRIP vectors) are able to transduce primary B and T cells, macrophages, dendritic cells, etc with a tenfold higher efficiency than other HIV vectors that lack the DNA flap. A transduction of 80-90% of cells can be routinely obtained.
  • a vector suitable for an in vivo expression and vaccine strategy is a retroviral and especially a lentiviral vector (see WO 99/55892 , WO 01/27300 and WO 01/27304 ).
  • a lentiviral vector see WO 99/55892 , WO 01/27300 and WO 01/27304 .
  • Such vectors have been shown to be particularly efficient and secure, when their genome is modified ( Firat et al. 2002, The Journal of Gene Medicine 4: 38-45 ). Indeed, these vectors have the ability to efficiently and stably transfer therapeutic or reporter genes, in a large variety of cells and tissues, such as hematopoietic stem cells, brain, liver and retina. Moreover, this high transduction efficiency is irrespective of the proliferative status of the target cells.
  • these vectors have been shown to efficiently induce CD8 + T cell responses both in vivo in mice and ex vivo in humans, due to their capacity to transduce antigen presenting cells such as dendritic cell (DC) with high efficiency, ex vivo as well as in vivo ( Esslinger et al. Hum Gene Ther 2002;13:1091-100 ; Breckpot et al. J Gene Med 2003;5:654-67 ; Esslinger et al. J Clin Invest 2003;111:1673-81 ).
  • DC dendritic cell
  • the vector has been used for in vitro or in vivo expression of epitopes, analogues or polyepitopes of the invention.
  • vectors from which the vectors of the invention can be derived, are the following, all deposited with the CNCM (Collection Nationale de Culture de Microorganismes at Institut Pasteur, Paris, France): Vector name Accession number Date of deposition Described in patent application PTRIP.EGFP I-2005 April 15, 1998 WO 99/55892 PTRIP-MEL.IRES-GFP I-2185 April 20, 1999 PTRIP-TEL/AML-IRES-GFP I-2326 October 11, 1999 WO 01/27300 PTRIP-TEL/ILKE-IRES-GFP I-2327 October 11, 1999 PTRIP-DES-IRES-GFP I-2331 October 11, 1999
  • the vectors described in the patent application WO 01/27304 can also be used to derive the vectors of the present invention.
  • a particular vector of the invention is the pTRIP-CMV- ⁇ hTERT vector, deposited at the CNCM (Institut Pasteur, Paris, France) under the number CNCM I-3660 on July 28, 2006.
  • a suitable growth medium for cultivating this vector is a TB medium, optionally supplemented with hygromycin.
  • Another expression vector of the invention is the pTRIP-CMV- ⁇ hTERT vector deposited at the CNCM under the number CNCM I-3660 on July 28, 2006, in which the deleted hTERT sequence has been substituted by any polynucleotide of the invention.
  • the present invention also relates to cells comprising the polynucleotides or polynucleotide units of the invention.
  • the cell is transfected with a vector of the invention, by methods well known to the man skilled in the art, i.e. by chemical transfection (calcium phospate, lipofectamine), lipid-based techniques (liposome), electroporation, photoporation, use of viral vectors....
  • a cell is transformed or transduced with a polynucleotide of the invention, in a way enabling integration of the polynucleotide in the cell genome either by a recombination with the homologous cellular sequence or by insertion in the cellular genome.
  • the transfection, infection or transduction can occur ex vivo, i.e. in an artificial environment outside the living organism.
  • cells of the immune system are cells of the immune system, and especially antigen presenting cells (APC).
  • APC antigen presenting cells
  • these cells are APCs involved either in MHC class I recognition, like dendritic cells (DC) or in MHC class II recognition such as macrophages or lymphocytes B.
  • DCs ex vivo fully maturated DCs, i.e., DCs that have been in vitro maturated by epitopes or analogues, are preferred.
  • transfected As used herein, the terms “transfected”, “transformed' or infected” refer to a cell comprising a vector of the invention (transient expression), whereas the term “genetically transformed” refers to a cell whose genome has been definitively modified by a polynucleotide of the invention (permanent expression).
  • Said transitory or stably transformed cells can be any prokaryotic (bacteria) or eukaryotic (yeast, animal including mammal especially human) cells.
  • cells are non-human cells.
  • cells of the invention are isolated human cells, "isolated" meaning outside of its natural environment.
  • a particular host is the E. coli strain deposited at the CNCM under the number CNCM I-3660 on July 28, 2006.
  • the invention also relates to epitopes, analogues or polyepitope as defined above when describing the polynucleotides of the invention and particularly to any polypeptide encoded by a polynucleotide or polynucleotide units of the invention.
  • Particular polypeptides are MHC class I-restricted, especially HLA-B7-restricted, hTERT epitope, chosen from the group consisting of:
  • HLA-B7-restricted hTERT epitopes are chosen from the group consisting of:
  • a particular group consists of the following MHC class I-restricted, especially HLA-B7-restricted, hTERT epitopes:
  • HLA-B7-restricted hTERT epitopes Another group consists of the following HLA-B7-restricted hTERT epitopes: RPAEEATSL (p277) and RPSFLLSSL (p342).
  • RPAEEATSL p277
  • RPSFLLSSL p342
  • a preferred HLA-B7 allele targeted by these epitopes is HLA-B0702.
  • the invention also concerns analogues of the peptides disclosed above, and having at least one amino acid substitution.
  • Analogues have especially been described in the above pages.
  • a particular peptide analogue is p444* having the following peptide sequence APRRLVQLL.
  • the invention also concerns any polynucleotide encoding a hTERT epitope, analogue or polyepitope as described in the present specification.
  • the invention also relates to a polyepitope comprising at least two epitopes and/or analogues as described above.
  • the polyepitopes of the invention are not the full-length hTERT protein.
  • the size of said polyepitope can range from 15 to 1000 in particular from 50 or from 100 to 1000 amino acids, especially and particularly about 100, 200, 300, 400, 500 or 1000 amino acids.
  • Such an epitope comprises or consists of 2 to 30 epitopes or analogues, and particularly 2 to 20 or 2 to 10 epitopes and/or analogues.
  • a particular polyepitope comprises or consists of 6 consecutive epitopes and has the following sequence: MPRAPRCRAAPRCRAVRSLAPSFRQVSCLR PAEEATSLRPSFLLSSLRPSLTGARRL.
  • Another particular polyepitope comprises or consists of the p1, p4, p68, p277, p342 and p351 epitopes, the epitopes being either consecutive to each other in the polyepitope obtained or all or part of them being separated by peptide spacers.
  • Another polyepitope of the invention comprises at least two epitopes, at least one being chosen from the group consisting of:
  • Polypeptides of the invention can be synthesized chemically, or produced either in vitro (cell free system) or in vivo after expression of the corresponding nucleic acid sequence in a cell system.
  • hTERT protein as represented in Figure 1 is excluded from the invention, as well as the corresponding full-length coding sequence. Also excluded from the present invention, the RPALLTSRL peptide. These peptides are excluded particularly in the context of HLA-B7 recognition.
  • the invention also concerns an epitope, analogue, polyepitope or polynucleotide, an expression vector or host cell as defined above, for use to elicit or participate in providing a HLA-B7-restricted immune response against hTERT.
  • the invention also concerns a composition
  • a composition comprising a polynucleotide, a vector, a host cell and/or a polypeptide of the invention.
  • said composition is suitable for in vivo administration, i.e., said composition is prepared for injection,or more generally for association with physiologically-acceptable liquid solutions or emulsions for administration.
  • Said composition may be used either for systemic or local administration, especially by injection, and may further comprises a pharmaceutically suitable excipient (including water, saline, dextrose, glycerol, ethanol, and combinations thereof) or a carrier and/or a vehicle.
  • said composition comprises a polynucleotide of the invention encoding an epitope, analogue or encoding a polyepitope as described above.
  • Said composition can comprise other nucleic acid molecules encoding at least one hTERT epitope or analogue thereof or polyepitope, restricted to a different MHC class I allele from that of HLA-B7.
  • the combination of hTERT epitopes restricted to different HLA supertypes or alleles enables covering a larger population of patients in need of treatment than a sole supertype or allele.
  • HLA-A1, -A2, -A3 and -A24 are preferred.
  • the composition comprises nucleic acid molecules encoding at least one hTERT epitope or analogue thereof or polyepitope, restricted to MHC class II.
  • Said composition can comprise any combination of nucleic acid molecules as described above, with at least one HLA-B7-restricted hTERT epitope, analogue or polyepitope of the present invention.
  • the combination, in a composition of nucleic acid molecules, of polynucleotides encoding Class I and Class II-restricted epitopes enabling the reaction of various immune cells (T lymphocytes or NK cells for class I versus helper lymphocytes for class II), and/or the elicitation of various immune responses (humoral versus cellular response) is also within the present invention.
  • the composition comprises nucleic acid molecules comprising at least such molecules encoding one tumour-specific antigen (TSA) and/or at least one tumour-associated antigen (TAA), such as prostate specific antigen (PSA), prostate-specific membrane antigen (PSMA) or prostatic acid phosphatase (PAP) ( Tartour et al. 2000 Immunol Lett Sep 15; 74(1): 1-3 ; Tartour et al. 1996 Presse Med. Nov 16; 25(25): 1717-22 ).
  • TSA tumour-specific antigen
  • TAA tumour-associated antigen
  • PSA prostate specific antigen
  • PSMA prostate-specific membrane antigen
  • PAP prostatic acid phosphatase
  • compositions can be used to elicit an immune response against an epitope or analogue of the invention.
  • a composition comprising a polynucleotide of the invention is administered to a host, for instance injected (known as DNA vaccination) and said nucleic acid expresses in vivo a polypeptide comprising or consisting of multiple epitopes according to the invention.
  • DNA vaccines usually consist of plasmid vectors as described above.
  • the delivery of naked DNA has shown to be poorly efficient, and some carriers are needed to improve the delivery and uptake of DNA into cells.
  • compositions comprising lentiviral pseudoparticles comprising a vector or vector genome as mentioned above.
  • compositions comprises an epitope, analogue or polyepitope of the invention.
  • a composition is immunogenic, i.e., it is capable of eliciting an immune response in a host in which it is administered.
  • an adjuvant can be administered with the polypeptide, to elicit or improve the immune response.
  • An adjuvant is defined as any substance that enhances the immunogenicity of an antigen mixed with said adjuvant.
  • adjuvants comprises sterile constituents of bacteria such as cell wall or polysaccharides, Freund adjuvant.
  • emulsifying agents or pH buffering agents can also be used to enhance the immunogenic behaviour of the epitope or analogue.
  • compositions quoted above can be injected in a host via different routes: subcutaneous (s.c.), intradermal (i.d.), intramuscular (i.m.) or intravenous (i.v.) injection, oral administration and mucosal administration, especially intranasal administration or inhalation.
  • the quantity to be administered depends on the subject to be treated, including the condition of the patient, the state of the individual's immune system, the route of administration and the size of the host. Suitable dosages range from 200 ⁇ g to 1mg, and can be modified by one skilled in the art, depending on circumstances.
  • compositions of the invention are useful for the prophylaxis and or treatment of malignant states in patients, resulting from uncontrolled cell proliferation, including tumors, resulting from the over-expression of hTERT, as well for the treatment of detrimental consequences accompanying such malignant state, e.g., cancer.
  • treatment encompasses the curative effect achieved with compositions of the invention and also the beneficial effect for the patient undergoing the treatment, said effect being either obtained at cellular level or clinical level, including as a result, an improvement of the condition of the patient and/or a remission state or a recovery of a health state.
  • the composition of the invention further comprises additional active compounds useful for the prophylaxis or the treatment of tumors, either general compounds or compounds proved to be active in a tissue-specific cancer.
  • the invention also concerns a process to activate T lymphocytes against class I-restricted, particularly HLA-B7-restricted, hTERT epitopes:
  • the invention also relates to a process to check the immunogenic behaviour of a hTERT peptide, comprising:
  • the epitope when used individually i.e., not under the form of a polyepitope is chosen among (a) RPSLTGARRL (p351),(b) APSFRQVSCL (p68), (c) APRCRAVRSL (p4), (d) DPRRLVQLL (p444), (e) FVRACLRRL (p464), (f) AGRNMRRKL (p966), (g) LPGTTLTAL (p1107) and (h) LPSPKFTIL (p1123); an example of analogue is the p444* as defined above.
  • a process to check the immunogenic behaviour and HLA-B7-restriction of a hTERT peptide comprising:
  • the activation of lymphocytes includes the presentation of said epitopes or analogues by antigen presenting cells to na ⁇ ve (not activated) T lymphocytes.
  • na ⁇ ve T lymphocytes Once na ⁇ ve T lymphocytes have recognized the epitope or analogue of the invention, in the context of a particular class-I HLA molecule, they are said "activated” and ready to recognize said epitope on the cell surface of a target cell.
  • the contact between said activated lymphocytes (effector cells) and target cells leads to the secretion of molecules and killing of the target cells.
  • an epitope or analogue used to activate lymphocytes leads to an efficient destruction of a target cell bearing said epitope by said activated lymphocytes
  • said epitope can be considered as immunogenic enough to allow not only in vitro but also in vivo T cell reaction against cells expressing said epitopes.
  • Suitable conditions for target cells/lymphocytes recognition are a 4 hour-contact at 37°C in RPMI medium.
  • the invention also relates to a process to in vitro maturate cells, and especially antigen presenting cells (APC), B cells, T cells and/or dendritic cells, against MHC class I-restricted, particularly HLA-B7-restricted, hTERT epitopes.
  • APC antigen presenting cells
  • B cells B cells
  • T cells T cells
  • dendritic cells against MHC class I-restricted, particularly HLA-B7-restricted, hTERT epitopes.
  • lymphocytes require epitope presentation by maturated antigen presenting cells.
  • said cells express at least one HLA-B7 allele.
  • dendritic cells are particularly efficient in presentation of endogenous epitopes restricted to MHC class I, to T lymphocytes.
  • One of the objectives in the maturation of said cells is their administration once maturated, to a patient in need of treatment. The administration of said maturated DCs would result in vivo in the activation of patient's lymphocytes, and rapid reaction against cell expressing the epitope (the one, the DCs have been transformed with).
  • a process to in vitro maturate dendritic cells comprises:
  • said dendritic cells are isolated from either circulating blood or bone marrow cells.
  • dendritic cells are isolated from the patient in need of treatment or from an HLA-matched donor, to avoid rejection after the administration to said patient.
  • the maturation of DCs can be achieved by genetic transformation of said dendritic cells with a polynucleotide of the invention, by transfection of said dendritic cells with a vector of the invention or by contacting said dendritic cells with at least one epitope, epitope analogue or polyepitope of the invention.
  • the genetic transformation is preferred because of its efficiency and the permanent expression of the epitope, analogue or polyepitope encoded by the polynucleotide inserted in the DC genome.
  • the invention also concerns a polynucleotide encoding a HLA-B7-restricted hTERT epitope for use in the prevention and/or treatment of cancer.
  • said polynucleotide for use in the prevention and/or treatment of cancer encodes a HLA-B7-restricted hTERT epitope or analogue thereof or a polyepitope comprising at least one HLA-B7-restricted hTERT epitope or analogue thereof as described in the present application.
  • the polyepitope-encoding polynucleotide does not coincide with the coding sequence of the full-length hTERT.
  • said polynucleotide comprises at least one polynucleotide unit encoding a HLA-B7-restricted hTERT epitope, chosen from the group consisting of:
  • the invention concerns a HLA-B7 hTERT epitope, and especially a HLA-B0702 restricted epitope for use in the prevention and/or treatment of cancer.
  • the invention relates also to a polynucleotide, a vector, a host cell or a polypeptide of the invention for use in the prevention and/or treatment of cancer.
  • the invention also relates to the use of a HLA-B7 hTERT epitope, (or corresponding polynucleotide) for the manufacture of a drug for the prevention and/or treatment of cancer.
  • HLA-B7 hTERT epitopes or corresponding polynucleotides
  • the use of polynucleotide, vector, host cell or polypeptide comprising or consisting of a polyepitope of the invention in the manufacture of a drug for the prevention and/or treatment of cancer is intended for patients having at least one HLA-B7 allele as defined above, and particularly at least one HLA-B0702 allele.
  • each definition provided in the specification applies to each and any peptide (epitope, analogue or polyepitope) as well as to each and any polynucleotide, taken individually (as such) or encompassed in a group.
  • Figure 1 Gene encoding the hTERT protein and corresponding amino acid sequence.
  • the coding sequence is located between the nucleotide 56 and 3454. Initiation and termination codons are underlined. First line is the nucleotide sequence; second line is the corresponding amino acid sequence. Third line is the numerotation of the hTERT coding sequence, starting from the initiation codon as the first amino acid.
  • FIG. 2 hTERT derived peptides are processed in HLA-B0702 transgenic mice.
  • HLA-B7 Tg mice and one na ⁇ ve mice were immunized with 100 ⁇ g DNA encoding Htert.
  • N na ⁇ ve mice
  • spleen cells from each mouse were separately in vitro stimulated with different hTERT-derived peptides.
  • Effector cells were essayed 6 days later against RMA-B7 targets loaded with relevant ( ⁇ ) or control ( ⁇ ) peptides as described in the material and methods. Percentage of lysis at a 60/1 ratio are shown (results from two independent experiments).
  • Figure 3 Induction of CTL response against hTERT in PBMC from health blood donors.
  • T-lymphocyte cells from HLA-*B0702 + healthy donors were activated with each of the six hTERT peptide-pulsed autologous PBMC as detailed in materials and Method. After four rounds of weekly stimulation, effector cells, pulsed with relevant ( ⁇ ) or control ( ⁇ ) peptides, were essayed for lytic activity against 51 Cr-labeled T2-B7 cells,. Percentage of lysis at a 20/1 effector-target ratio is shown. Results from 8 out of 10 donors are presented (d1 to d8).
  • the cytotoxicity of the CTLp351 line against HLA-*B0702 + tumor cell lines Mamo and U293T pre-treated either in absence (none) or presence of HLA mAbs (anti-HLA class I mAb or an anti-class II mAb (HLA-DR)) was determined by standard 51 Cr-release assay at effector-target ratio of 10/1.
  • HLA-B7 transgenic mice were immunized with recombinant Trip-hTERT particles or control Trip-GFP (1500ng). After 12 days, hTERT peptide-specific T cells producing IFN ⁇ of each mouse were detected ex vivo by IFN ⁇ -ELISPOT assay within freshly spleen cells. The number of IFN ⁇ SFCs was calculated after subtracting negative control values. Results from three independent experiments are represented.
  • HHD mice were immunized with Trip-hTERT as described above.
  • hTERT peptide-specific T cells producing IFN ⁇ were detected ex vivo by ELISPOT as described above. Results from two independent experiments are represented.
  • HLA-B*0702 transgenic mice were immunized either with Trip-hTERT (4 first black bars) or control (2 last horizontal vertical stripe bars). 12 days later, IFN- ⁇ producing-single cells within splenocytes of each mouse were detected ex vivo by IFN- ⁇ ELISPOT assay. Ficoll purified lymphocytes from freshly isolated splenocytes of individual immunized mice were directly cultured with or without 5 ⁇ g/ml of each HLA-B*0702-restricted hTERT-derived peptides for 24h. The number of specific-IFN- ⁇ SFCs was calculated after subtracting non-specific values obtained with control without peptide ( ⁇ 15 SFC), and the responses were considered positive for SFC ⁇ 30.
  • This lentiviral-derived vector contains the psi sequence, the cPPT and CTS central cis-active sequences (Flap) of the HIV-1 genome and the CMV promoter which allows the expression of the gene of interest. Moreover, the U3 domain is deleted in the 3'LTR ( ⁇ U3).
  • HHD mice HLA-A2.1 Tg
  • pTRIP-CMV- ⁇ hTERT DNA immunized with a DNA encoding a non-functional form of HTERT
  • IFN- ⁇ producing peptide-specific T cells were detected ex vivo by IFN- ⁇ -ELISPOT assay.
  • Ficoll purified lymphocytes from splenocytes of individual immunized mouse were directly cultured for 24h, with or without 5 ⁇ g/ml of each HLA-A2.1-restricted hTERT-derived peptide. The number of specific- IFN- ⁇ SFCs was calculated as described above. Responses were considered positive for SFC ⁇ 30.
  • HHD mice were immunized with a DNA encoding a non-functional form of HTERT (pTRIP-CMV- ⁇ hTERT) for 10 days.
  • Spleen cells from individual mice were restimulated in vitro with HLA-A2.1-restricted hTERT-derived p540 and pY572 peptides for 6 days. Effector cells were tested in a 51 Cr-release assay against HHD-transfected EL4 cells loaded with either the relevant peptide or the irrelevant peptide.
  • Figure 9 Sequence of a non-functional hTERT protein (deletion of amino acids 867 to 869)
  • the coding sequence is located between the nucleotide 59 and 3348. Initiation and termination codons are underlined. First line is the nucleotide sequence; second line is the corresponding amino acid sequence. Third line is the numerotation of the hTERT coding sequence, starting from the initiation codon as the first amino acid.
  • Figure 10 Sequence of a non-functional hTERT protein (deletion of amino acids 864 to 872)
  • the coding sequence is located between the nucleotide 59 and 3430. Initiation and termination codons are underlined. First line is the nucleotide sequence; second line is the corresponding amino acid sequence. Third line is the numerotation of the hTERT coding sequence, starting from the initiation codon as the first amino acid.
  • Peripheral bloods samples were obtained following written informed consent from adult healthy platelet donors (centre de transfusion sanguine de l'sufficient Mondor, Cruteil, France). HLA typing of peripheral blood donors was performed in the HLA laboratory of the H. Mondor. Hospital Creteil (France). The study was approved by the French Blood Bank Institute.
  • HLA-*B0702 transgenic mice expressing an HLA-B0702 ⁇ 1 ⁇ 2, H2-Kd ⁇ 3 chimeric construct, in combination with constitutive murine ⁇ 2-m molecule (HLA-B7 m ⁇ 3 ) and HHD transgenic mice expressing a chimeric HLA-A2.1/H2-Db molecule, were deleted of their H2-Db and H2-k b genes as previously described ( Pascolo et al. J Exp Med 1997;185 :2043-51 ; Rohrlich et al. Int Immunol 2003;15:765-72 ). These mice are on a C57BL/6 background and were bred and maintained under specific pathogen-free conditions in our animal facility.
  • T-B hybrid T1 EBV-transformed B cell JY
  • renal cancer cell line U293T and Burkitt lymphoma cell Raji were from American type Culture Collection (ATCC).
  • Melanoma cell lines (SK23MEL, LB34, and KUL68) were kindly provided by P. Coulie (Bruxell, Belgium) and EBV-transformed B cell BBG.1 and BC3 were kindly provided by H. Collandre (R.A.H.P., Grenoble, France).
  • HLA-*B0702 transfected TAP deficient T2 cells were kindly provided by P. Cresswell ( Smith et al. J Immunol 1996;156:3755-64 ).
  • Murine lymphoma cell lines RMA, and EL4 were from ATCC; theses cells were also transfected with HLA-*B0702 gene and used as target cells
  • Peptides derived from human cytomegalovirus pp65, R P HERNGFT V (R10TV), and human immunodeficiency virus type 1 IPRRIRQGL were synthesized and were used as control peptides.
  • Peptide derived from hepatitis B virus core 128-140 (TPPATRPPNAPIL) was used as helper peptide for peptide immunization in mice.
  • Peptides were purchased from PRIM to a minimum purity 80% and were reconstituted in distilled water or DMSO at a concentration of 2mg/ml.
  • the relative avidity of hTERT derived peptides for HLA-*B0702 was measured by using a MHC stabilization assay on HLAB0702 transfected T2 (T2-B7) cells in comparison with a reference peptide (R10TV) as described ( Rohrlich et al. Int Immunol 2003;15:765-72 ).
  • T2-B7 were incubated overnight at 2 x 10 5 cells/well in 96-well plates in serum free medium AIM-V (Invitrogen Corp., Gibco), supplemented with 100ng/ml of human ⁇ 2-microglobulin, in the absence (negative control) or in the presence of either reference peptide R10V or hTERT peptides at various final concentrations (100, 10, 1 and 0.1 ⁇ M).
  • T2-B7 cells were labelled with a saturating concentration of ME.1 an anti-HLA-B7 mAb, then washed twice and finally stained with FITC-conjugated F(ab')2 goat anti-mouse Ig before flow cytometry.
  • Results are expressed as values of relative avidity, that is the ratio of concentration of test peptide necessary to reach 20% of the maximal binding (obtained with the reference peptide) over the concentration of the reference peptide. Therefore, the lower the value, the stronger the binding.
  • mice at 8-10 weeks of age were injected subcutaneously (s.c.) at the base of the tail with 50 ⁇ g of individual HLA-B0702 restricted hTERT peptides supplemented with 140 ⁇ g of helper peptide co-emulsified in incomplete Freund's adjuvant (Difco, Detroit, MI).
  • spleen cells of individual mouse were re-activated in vitro with relevant peptide in six wells plate. Effector CTL cells were tested in a standard 4-5 h 51 Cr-release assay, using relevant or negative control peptide-pulsed, HLA-*B0702 transfected RMA cells (RMA-B7). Mice were considered as responders, when specific lysis ⁇ 10% was observed.
  • the LvCMV-hTERT plasmid vector encoding the hTERT gene under the control of CMV promotor was purified on plasmid Giga kit columns under endotoxin-free conditions (Qiagen).
  • Anesthetized HLA-*B0702 Transgenic mice were injected with said plasmid (50 ⁇ g each side) into regenerating tibialis anterior muscles. 14 days after, spleen cells of individual mouse were re-activated in vitro with peptide-pulsed (10 ⁇ g/ml), syngenic ⁇ -irradiated (50 Gy) LPS-lymphoblast in complete medium, supplemented with 10% supernatant from Con A-activated rat spleens cells. Cytotoxicity assays were performed 6 days as described.
  • the pTRIP-deltaU3-CMV-hTERT (referred as TRIPLv-hTERT or Lv-hTERT or pTrip-hTERT) ( Figure 7 ) construct was created by first subcloning an EcoRI-Sall hTERT insert derived from the pBABE-hygro-hTERT plasmid ( Counter et al. Proc Natl Acad Sci U.S.A. 1998;95:14723-8 ) into the pSP73 vector (Promega). A BglII-SalI fragment was then inserted into the pTRIP-CMV plasmid cut with BamH1 and Xhol.
  • Pseudo typed recombinant retroviral particles were produced by transient (48h) transfection of 293T cells as described ( Zennou et al.. Cell 2000;14;101:173 ; Firat et al. J Gene Med 2002;4:38-45 ).
  • the recombinant retroviral particles were concentrated by ultra-centrifugation and resuspended in PBS.
  • the amount of vector particles was estimated from that of p24 protein in a commercially available ELISA assay (NEN, DUPONT, France Perkin Elmer).
  • the pTRIP-CMV- ⁇ hTERT vector deposited at the CNCM (Institut Pasteur, Paris, France) under the number CNCM I-3660 on July 28, 2006, was carried out as described in the paragraph above.
  • the hTERT protein was rendered non-functional by deletion of amino acids 867 to 869, corresponding to nucleotides 2654 to 2662 of Figure 1 (wild-type).
  • the catalytically dead hTERT RT mutant ( ⁇ hTERT) was generated by creating a deletion of amino acid residues 867 to 869 using the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene) and verified by sequencing.
  • Immunization with TRIPLv-hTERT was performed as a single subcutaneously (at the base of the tail) injection of 1500 ng of TripLv-hTERT suspension or control vector.
  • Immunization was performed in HLA.A2 transgenic mice as a single intraperitoneal injection of recombinant lentiviral particles, pTRIP-CMV- ⁇ hTERT or Trip-GFP as a control, equivalent to 1500 ng of p24 antigen in 500 ⁇ l of PBS.
  • hTERT peptide-specific T among splenocytes were detected by an ELISPOT assay (see below). Cytotoxicity assays were performed on the same immune splenocyte populations after in vitro stimulation with peptide-pulsed as described above.
  • Peptide-specific T cells from immunized mice were detected by IFN- ⁇ ELISPOT assay as previously described ( Miyahira et al. J Immunol Methods 1995;181:45-54 ).
  • Anti-mouse IFN- ⁇ mAb's (3 ⁇ g/ml; Pharmigen, Becton Dickinson biosciences) were coated onto 96-well nitrocellulose microplates (multi screen; Millipore corp, Molsheim, France). After red cell lysis, freshly isolate spleen lymphocytes of individual mouse (5x10 5 , 2.5x10 5 and 1.25x10 5 cells/well) were directly cultured with or without 5 ⁇ g of native hTERT peptide for 18h at 37°C.
  • IFN ⁇ spot-forming cells were developed by adding peroxidase substrates (BCIP/NBT, Promega Corp, Madison W; USA) and counted using automated image analysis system a Bioreader 2000 (Biosys, Karben, germany). The number of specific SFCs was calculated after subtracting negative control values ( ⁇ 10 SFC). Responses were positive if the mean of SFCs in stimulated well was greater than the mean + 2 S.D. of the SFCs in the negative control wells and greater than 50 SFC/10 6 cells.
  • Cytotoxicity assays were performed by using standard 4-5h 51 Cr release assay as previously described ( Firat et al. J Gene Med 2002;4:38-45 ). Specific lysis in % was calculated by subtracting non-specific lysis observed with the control peptide. Mice were considered as responsers when specific lysis ⁇ 10% was observed.
  • lymphocytes were re-activated with peptide-pulsed ⁇ -irradiated autologous PBMCs (50 Gy). The next day, 20 IU/ml human IL-2 (Roche, Mannheim, Germany) was added to the culture. CTL lines were re-activated weekly during four cycles. For some donors, CD8 + T cells were purified after three round cycle, using CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany.) according to manufacturer's recommendations and activated once before functional test. Cytolytic assays were performed 6 days after the last re-activation against various 51 Cr-labeled targets: T2-B7 pulsed with tested hTERT peptides or irrelevant peptide, or tumor cell lines.
  • tumors cells were incubated with an anti-HLA class I framework mAb, w6/32 (BD Pharmigen) or anti-HLA-*B0702 mAb, ME.1, or an anti-HLA-DR mAbB , G46.6 (BD Pharmigen) at an optimal concentration (10 ⁇ g/mL) for 30 minutes to determine whether cytotoxicity was restricted to HLA class I.
  • an anti-HLA class I framework mAb, w6/32 (BD Pharmigen) or anti-HLA-*B0702 mAb, ME.1, or an anti-HLA-DR mAbB , G46.6 (BD Pharmigen) at an optimal concentration (10 ⁇ g/mL) for 30 minutes to determine whether cytotoxicity was restricted to HLA class I.
  • hTERT-derived peptides were processed in HLA-B0702 transgenic mice.
  • HLA-*B0702 transgenic mice were immunised with cDNA encoding hTERT, forty days after peptide-specific CTL responses within spleen cells of individual mice were evaluated.
  • hTERT peptide-specific CTLs were induced in most immunized mice (M), from 50 to 80% of mice for p4, p68, p1, p277and p351.
  • M immunized mice
  • p342-specific CTLs can be induced in about 15% tested mice.
  • hTERT-specific CTLs from donors were tested for their capacity to lyse human tumour cell lines of different origins.
  • the results presented in Table 2 show that, CTL lines generated in vitro from healthy donors killed HLA-B0702 + tumour cells, whereas no cytotoxicity against HLA-B0702 - tumors was detected.
  • CTLp351 in d 1 , d 2 and d 3 in KU L268 or 293-UT target (respectively 52, 25, 20 and 34, 41 and 19%) versus T1 or BBG1 target (respectively 9, 2, 6 and 0, 0, 2 %).
  • Differences were observed in tumor recognition according to the CTL specificity; this could be explained by differential presentation of hTERT peptides on the surface of the tumor cells.
  • p351-specific CTL lines generated from different donors recognize the majority of tumor cell lines tested (Table 2). In contrast, all p4-specific CTL lines do not lyse all the type of tested tumors. CTL lines, specific for p1 and p68 peptides, only recognize the T1-B7 targets. p342-specific CTL lines recognize only melanoma cells (LB 34 and KU 268 target). Finally, p277-specific CTL lines recognize renal cancer (293 UT) but neither melanoma nor lymphoid tumor cells. On the other hand, normal PBMCs and CD40 activated B cells were not lysed by these hTERT peptide-specific CTL lines, regardless of HLA type (two last lines of Table 2).
  • Lentiviral vector encoding hTERT vaccination induces efficient peptide-specific T cell responses in mice.
  • peptide-specific CD8 + T cell responses were obtained against HLA-B0702 restricted hTERT epitopes, as compared with mice that received Lv-GFP control vector.
  • Functional analysis of the induced peptide-specific CD8 + cells in chromium release assay after in vitro stimulation confirmed ex vivo ELISPOT data (Table 3) and show that efficient specific CTL response is generated against these six peptides in about 50-70 % of mice after a single injection of Lv-hTERT and in 100% of the mice after a boost with TRIPLv-hTERT ( figure 6 ). This was also associated with strong CTL responses in all mice (Table 3 ).
  • hTERT peptide specific-CTL lines (CTLp1, CTLp4, CTLp68, CTLp277, CTLp342, CTLp351) were obtained from healthy donors that were responder after subsequent in vitro immunization as described in material and methods. Cytotoxicity was measured in a standard 51 Cr-labeled release assay. Specific lysis: for a 30:1 effector: target ratio were shown ⁇ . Autologous B lymphocytes from normal donors were activated for 48h with a trimeric CD40 L (40 ⁇ g/ml).
  • hTERT epitopes which are in vivo immunogenic and processed in H-2-class I knockout HLA-B0702 transgenic mice have identified. Further, in vitro, hTERT peptide immunization using HLA-B702 + PBL from healthy donors induce specific CTL responses recognizing hTERT + tumors from various origins, implying that there is no deletion in the human T cell repertoire for these epitopes. Moreover, it was shown that depending upon the tumor origins, peptides repertoire expressed on the cell surface could be qualitatively different, underlining, the utility to characterize hTERT as polyepitope tumor associated antigens for circumvent antigenic variability of cancer cells.
  • HLA-*B0702 and HLA-A2 1 transgenic mice were used, to test a candidate vaccine consisting of a non-functional telomerase gene inserted in a new generation of lentiviral derived flap vector.
  • a strong hTERT specific CD8 + T cell responses were observed in all the HLA-transgenic mice.
  • telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes.Immunity. 1999 Jun;10(6):673-9 .
  • H-2 class I knockout HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies. Eur J Immunol. 1999 Oct;29(10):3112-21 .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
EP13165854.4A 2005-07-29 2006-07-31 Polynucleotide zur Codierung von MHC Class I HLA-B7-beschränkten HTERT-Epitopen, Analoga davon oder Polyepitope Active EP2626420B1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP13165854.4A EP2626420B1 (de) 2005-07-29 2006-07-31 Polynucleotide zur Codierung von MHC Class I HLA-B7-beschränkten HTERT-Epitopen, Analoga davon oder Polyepitope

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP05291627A EP1748067A1 (de) 2005-07-29 2005-07-29 Polynukleotide, die für MHC Klasse I-beschränkte Epitope der hTERT, Analoge und Polyepitope kodieren
EP06762910.5A EP1910529B1 (de) 2005-07-29 2006-07-31 Für hla-b7-restringierte htert-epitope codierende polynukleotide zur anwendung in der prevention und/oder behandlung von krebs
EP13165854.4A EP2626420B1 (de) 2005-07-29 2006-07-31 Polynucleotide zur Codierung von MHC Class I HLA-B7-beschränkten HTERT-Epitopen, Analoga davon oder Polyepitope

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
EP06762910.5 Division 2006-07-31
EP06762910.5A Division EP1910529B1 (de) 2005-07-29 2006-07-31 Für hla-b7-restringierte htert-epitope codierende polynukleotide zur anwendung in der prevention und/oder behandlung von krebs

Publications (3)

Publication Number Publication Date
EP2626420A2 true EP2626420A2 (de) 2013-08-14
EP2626420A3 EP2626420A3 (de) 2013-10-30
EP2626420B1 EP2626420B1 (de) 2016-06-22

Family

ID=35735256

Family Applications (3)

Application Number Title Priority Date Filing Date
EP05291627A Ceased EP1748067A1 (de) 2005-07-29 2005-07-29 Polynukleotide, die für MHC Klasse I-beschränkte Epitope der hTERT, Analoge und Polyepitope kodieren
EP06762910.5A Active EP1910529B1 (de) 2005-07-29 2006-07-31 Für hla-b7-restringierte htert-epitope codierende polynukleotide zur anwendung in der prevention und/oder behandlung von krebs
EP13165854.4A Active EP2626420B1 (de) 2005-07-29 2006-07-31 Polynucleotide zur Codierung von MHC Class I HLA-B7-beschränkten HTERT-Epitopen, Analoga davon oder Polyepitope

Family Applications Before (2)

Application Number Title Priority Date Filing Date
EP05291627A Ceased EP1748067A1 (de) 2005-07-29 2005-07-29 Polynukleotide, die für MHC Klasse I-beschränkte Epitope der hTERT, Analoge und Polyepitope kodieren
EP06762910.5A Active EP1910529B1 (de) 2005-07-29 2006-07-31 Für hla-b7-restringierte htert-epitope codierende polynukleotide zur anwendung in der prevention und/oder behandlung von krebs

Country Status (9)

Country Link
US (3) US8858931B2 (de)
EP (3) EP1748067A1 (de)
JP (4) JP5631545B2 (de)
CA (1) CA2616674C (de)
DK (2) DK1910529T3 (de)
ES (2) ES2440768T3 (de)
PL (1) PL2626420T3 (de)
PT (1) PT2626420T (de)
WO (1) WO2007014740A2 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10383928B2 (en) 2010-02-16 2019-08-20 Ultimovacs As Telomerase polypeptide vaccine for treating cancer

Families Citing this family (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1748067A1 (de) * 2005-07-29 2007-01-31 Institut Pasteur Polynukleotide, die für MHC Klasse I-beschränkte Epitope der hTERT, Analoge und Polyepitope kodieren
US10767167B2 (en) 2005-07-29 2020-09-08 Institut Pasteur Polynucleotides encoding MHC class I-restricted hTERT epitopes, analogues thereof or polyepitopes
WO2008010010A1 (en) * 2006-07-12 2008-01-24 Vaxon Biotech Identification, optimization and use of cryptic hla-b7 epitopes for immunotherapy
US20080171062A1 (en) * 2006-08-16 2008-07-17 Monica Sala-Schaeffer Recombinant HBsAg virus-like particles containing polyepitopes of interest, their production and use
US8188214B2 (en) * 2007-02-28 2012-05-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Brachyury polypeptides and methods for use
EP2565204B1 (de) * 2007-07-27 2015-10-07 immatics biotechnologies GmbH Neuartige immunogenetische Epitope zur Immuntherapie
US7904566B2 (en) * 2008-03-14 2011-03-08 Silicon Image, Inc. Method, apparatus, and system for employing an enhanced port multiplier
US7979589B2 (en) * 2008-03-14 2011-07-12 Silicon Image, Inc. Method, apparatus, and system for port multiplier enhancement
CA2865553A1 (en) 2012-03-15 2013-09-19 Macrocure, Ltd. Activated immunostimulatory cell composition and uses thereof
CA2864838A1 (en) 2012-03-15 2013-09-19 Macrocure, Ltd. Activated immunostimulatory cell composition for therapy of infection
EP2639299A1 (de) 2012-03-16 2013-09-18 Invectys Aus Telomerase abgeleitete, universelle Krebspeptide
WO2014105870A1 (en) 2012-12-27 2014-07-03 Sierra Sciences, Inc. Enhancing health in mammals using telomerase reverse transcriptase gene therapy
AU2014242915B2 (en) 2013-03-28 2018-08-09 Invectys A cancer vaccine for cats
JP6479761B2 (ja) * 2013-03-28 2019-03-06 アンヴェクティＩｎｖｅｃｔｙｓ イヌ用がんワクチン
GB201315946D0 (en) * 2013-09-06 2013-10-23 Immune Targeting Systems Its Ltd Oncology vaccine
EP3062824B1 (de) 2013-10-28 2019-11-27 Invectys Dna-impfstoff zur telomerasecodierung
ES2753412T3 (es) 2013-10-28 2020-04-08 Invectys Electrotransferencia génica a las células de la piel
AU2016206682B2 (en) 2015-01-14 2021-11-11 The Brigham And Women's Hospital, Inc. Treatment of cancer with anti-LAP monoclonal antibodies
US11254914B2 (en) 2015-03-12 2022-02-22 Health Research, Inc. Enrichment of CD16+ monocytes to improve dendritic cell vaccine quality
EP3283529B1 (de) 2015-04-17 2023-06-07 The General Hospital Corporation Wirkstoffe, systeme und verfahren zur behandlung von krebs
PT3359183T (pt) * 2015-10-06 2020-08-13 Invectys Construções de poliepítopo para utilização em imunoterapia
GB201519557D0 (en) 2015-11-05 2015-12-23 Univ Witwatersrand Jhb Compounds for use in the treatment of telomere related diseases and/or telomere related medical conditions
ES2811523T3 (es) 2016-01-19 2021-03-12 Pfizer Vacunas contra el cáncer
MX2019003722A (es) * 2016-09-30 2019-09-26 Univ Pennsylvania Composiciones inmunogénicas de tert y métodos de tratamiento que las utilizan.
EP3622078A1 (de) 2017-05-09 2020-03-18 Invectys Rekombinanter masernimpfstoff mit htert-expression
AU2018395397A1 (en) * 2017-12-28 2020-08-06 Gritstone Bio, Inc. Antigen-binding proteins targeting shared antigens
CN110305217B (zh) 2018-03-27 2022-03-29 广州爱思迈生物医药科技有限公司 双特异性抗体及其应用
WO2019197610A1 (en) 2018-04-13 2019-10-17 INSERM (Institut National de la Santé et de la Recherche Médicale) New vaccinal strategy
CN113105528B (zh) * 2020-01-13 2023-12-08 辽宁中健医药科技有限公司 一条肿瘤相关抗原ctl表位肽及其应用
EP4171619A2 (de) * 2020-06-26 2023-05-03 National Breast Cancer Coalition Brustkrebsimpfstoff

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055892A1 (fr) 1998-04-24 1999-11-04 Institut Pasteur Utilisation de sequences d'adn de structure triplex pour le transfert de sequences nucleotidiques
WO2000002581A1 (en) 1998-07-08 2000-01-20 Norsk Hydro Asa Antigenic peptides derived from telomerase
WO2001027300A1 (en) 1999-10-11 2001-04-19 Institut Pasteur Lentiviral vectors for the preparation of immunotherapeutical compositions
WO2001027304A2 (en) 1999-10-12 2001-04-19 Institut Pasteur Lentiviral triplex dna, and vectors and recombinant cells containing lentiviral triplex dna

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE245194T1 (de) * 1996-10-01 2003-08-15 Geron Corp Katalytische untereinheit der menschlichen telomerase
US7622549B2 (en) * 1997-04-18 2009-11-24 Geron Corporation Human telomerase reverse transcriptase polypeptides
US7413864B2 (en) * 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
IL133830A0 (en) * 1997-07-01 2001-04-30 Cambia Biosystems Llc Vertebrate tolomerase genes and proteins and uses thereof
JP2002509716A (ja) * 1998-03-31 2002-04-02 ユニバーシティ テクノロジー コーポレイション テロメラーゼ抗原に対する免疫応答を惹起するための方法および組成物
FR2862648B1 (fr) * 2003-11-21 2006-02-03 Biomerieux Sa Nouveau peptide immunogene et nouveaux epitopes et utilisations notamment dans la preparation de compositions pharmaceutiques actives contre le virus de l 'hepatite c
CA2504451A1 (en) * 2004-08-10 2006-02-10 Geron Corporation Dendritic cell vaccines for treating cancer made from embryonic stem cells
EP1748067A1 (de) * 2005-07-29 2007-01-31 Institut Pasteur Polynukleotide, die für MHC Klasse I-beschränkte Epitope der hTERT, Analoge und Polyepitope kodieren

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999055892A1 (fr) 1998-04-24 1999-11-04 Institut Pasteur Utilisation de sequences d'adn de structure triplex pour le transfert de sequences nucleotidiques
WO2000002581A1 (en) 1998-07-08 2000-01-20 Norsk Hydro Asa Antigenic peptides derived from telomerase
WO2001027300A1 (en) 1999-10-11 2001-04-19 Institut Pasteur Lentiviral vectors for the preparation of immunotherapeutical compositions
WO2001027304A2 (en) 1999-10-12 2001-04-19 Institut Pasteur Lentiviral triplex dna, and vectors and recombinant cells containing lentiviral triplex dna

Non-Patent Citations (49)

* Cited by examiner, † Cited by third party
Title
ARAI ET AL., BLOOD, vol. 9, 2001, pages 2903 - 7
ARAI J; YASUKAWA M; OHMINAMI H; KAKIMOTO M; HASEGAWA A; FUJITA S.: "Identification of human telomerase reverse transcriptase- derived peptides that induce HLA-A24-restricted antileukemia cytotoxic T lymphocytes", BLOOD, vol. 97, no. 9, 1 May 2001 (2001-05-01), pages 2903 - 7
AYYOUB M; MIGLIACCIO M; GUILLAUME P; LIENARD D; CEROTTINI JC; ROMERO P; LEVY F; SPEISER DE; VALMORI D.: "Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing", EUR J IMMUNOL., vol. 31, no. 9, September 2001 (2001-09-01), pages 2642 - 51
BRECKPOT ET AL., J GENE MED, vol. 5, 2003, pages 654 - 67
BRECKPOT K; DULLAERS M; BONEHILL A; VAN MEIRVENNE S; HEIRMAN C; DE GREEF C; VAN DER BRUGGEN P; THIELEMANS K.: "Lentivirally transduced dendritic cells as a tool for cancer immunotherapy", J GENE MED., vol. 5, no. 8, August 2003 (2003-08-01), pages 654 - 67
COUNTER ET AL., BLOOD, vol. 85, 1995, pages 2315 - 20
COUNTER ET AL., PROC NATL ACAD SCI U.S.A., vol. 95, 1998, pages 14723 - 8
ESSLINGER C; CHAPATTE L; FINKE D; MICONNET , GUILLAUME P; LEVY F; MACDONALD HR: "In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8(+) T cell responses", J CLIN INVEST., vol. 111, no. 11, June 2003 (2003-06-01), pages 1673 - 81
ESSLINGER C; ROMERO P; MACDONALD HR: "Efficient transduction of dendritic cells and induction of a T-cell response by third- generation lentivectors", HUM GENE THER., vol. 13, no. 9, 10 June 2002 (2002-06-10), pages 1091 - 100
ESSLINGER ET AL., HUM GENE THER, vol. 13, 2002, pages 1091 - 100
ESSLINGER ET AL., J CLIN INVEST, vol. 111, 2003, pages 1673 - 81
EUR. J. OF IMMUNOL., vol. 29, 1999, pages 3112 - 3121
FIRAT ET AL., EUR. J. OF IMMUNOL., vol. 29, 1999, pages 3112 - 3121
FIRAT ET AL., J GENE MED, vol. 4, 2002, pages 38 - 45
FIRAT ET AL., THE JOURNAL OF GENE MEDICINE, vol. 4, 2002, pages 38 - 45
FIRAT H; GARCIA-PONS F; TOURDOT S; PASCOLO S; SCARDINO A; GARCIA Z; MICHEL ML; JACK RW; JUNG G; KOSMATOPOULOS K: "H-2 class I knockout, HLA-A2.1-transgenic mice: a versatile animal model for preclinical evaluation of antitumor immunotherapeutic strategies", EUR J IMMUNOL., vol. 29, no. 10, October 1999 (1999-10-01), pages 3112 - 21
FIRAT H; ZENNOU V; GARCIA-PONS F; GINHOUX F; COCHET M; DANOS 0; LEMONNIER FA; LANGLADE-DEMOYEN P; CHARNEAU P.: "Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy", J GENE MED., vol. 4, no. 1, January 2002 (2002-01-01), pages 38 - 45
FROLKIS M; FISCHER MB; WANG Z; LEBKOWSKI JS; CHIU CP; MAJUMDAR AS: "Dendritic cells reconstituted with human telomerase gene induce potent cytotoxic T-cell response against different types of tumors", CANCER GENE THER., vol. 10, no. 3, March 2003 (2003-03-01), pages 239 - 49
GROSS DA; GRAFF-DUBOIS S; OPOLON P; CORNET S; ALVES P; BENNACEUR-GRISCELLI A; FAURE 0; GUILLAUME P; FIRAT H; CHOUAIB S: "High vaccination efficiency of low-affinity epitopes in antitumor immunotherapy", J CLIN INVEST., vol. 113, no. 3, February 2004 (2004-02-01), pages 425 - 33
HAHN ET AL., NAT MED, vol. 5, 1999, pages 1164 - 70
HEISER A; MAURICE MA; YANCEY DR; COLEMAN DM; DAHM P; VIEWEG J.: "Human dendritic cells transfected with renal tumor RNA stimulate polyclonal T-cell responses against antigens expressed by primary and metastatic tumors", CANCER RES., vol. 61, no. 8, 15 April 2001 (2001-04-15), pages 3388 - 93
HERNANDEZ ET AL., PROC NATL ACAD SCI U.S.A., vol. 99, 2002, pages 12275 - 80
HERNANDEZ J; GARCIA-PONS F; LONE YC; FIRAT H; SCHMIDT JD; LANGLADE-DEMOYEN P; ZANETTI M.: "Identification of a human telomerase reverse transcriptase peptide of low affinity for HLA A2.1 that induces cytotoxic T lymphocytes and mediates lysis of tumor cells", PROC NATL ACAD SCI U S A., vol. 99, no. 19, 6 September 2002 (2002-09-06), pages 12275 - 80
LU; CELIS, E CANCER RES, vol. 60, 2000, pages 5223 - 7
MINEV B; HIPP J; FIRAT H; SCHMIDT JD; LANGLADE-DEMOYEN P; ZANETTI M.: "Cytotoxic T cell immunity against telomerase reverse transcriptase in humans", PROC NATL ACAD SCI U S A., vol. 97, no. 9, 25 April 2000 (2000-04-25), pages 4796 - 801
MINEV ET AL., PNAS, vol. 97, no. 9, 2000, pages 4796 - 4801
MIYAHIRA ET AL., J IMMUNOL METHODS, vol. 181, 1995, pages 45 - 54
PARKHURST ET AL., CLIN CANCER RES, vol. 10, 2004, pages 4688 - 98
PARKHURST MR; RILEY JP; IGARASHI T; LI Y; ROBBINS PF; ROSENBERG SA.: "Immunization of patients with the hTERT:540-548 peptide induces peptide-reactive T lymphocytes that do not recognize tumors endogenously expressing telomerase.", CLIN CANCER RES., vol. 10, no. 14, 15 July 2004 (2004-07-15), pages 4688 - 98
PASCOLO ET AL., J EXP MED, vol. 185, 1997, pages 2043 - 51
ROHRLICH ET AL., INT IMMUNOL, vol. 15, 2003, pages 765 - 72
ROHRLICH PS; CARDINAUD S; LULE J; MONTERO-JULIAN FA; PRODHOMME V; FIRAT H; DAVIGNON JL; PERRET E; MONSEAUX S; NECKER A: "Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-Transgenic mice and for ex vivo stimulation and expansion of CD8(+) cytotoxic T cells from human peripheral blood cells", HUM IMMUNOL., vol. 65, no. 5, May 2004 (2004-05-01), pages 514 - 22
SCARDINO A; GROSS DA; ALVES P; SCHULTZE JL; GRAFF-DUBOIS S; FAURE 0; TOURDOT S; CHOUAIB S; NADLER LM; LEMONNIER FA: "HER-2/neu and hTERT cryptic epitopes as novel targets for broad spectrum tumor immunotherapy", J IMMUNOL., vol. 168, no. 11, 1 June 2002 (2002-06-01), pages 5900 - 6
SCHROERS R; HUANG XF; HAMMER J; ZHANG J; CHEN SY: "Identification of HLA DR7-restricted epitopes from human telomerase reverse transcriptase recognized by CD4+ T-helper cells", CANCER RES., vol. 62, no. 9, 1 May 2002 (2002-05-01), pages 2600 - 5
SMITH ET AL., J IMMUNOL, vol. 156, 1996, pages 3755 - 64
SU Z; VIEWEG J; WEIZER AZ; DAHM P; YANCEY D; TURAGA V; HIGGINS J; BOCZKOWSKI D; GILBOA E; DANNULL J.: "Enhanced induction of telomerase-specific CD4(+) T cells using dendritic cells transfected with RNA encoding a chimeric gene product", CANCER RES., vol. 62, no. 17, 1 September 2002 (2002-09-01), pages 5041 - 8
SUN B; HUANG Q; LIU S; CHEN M; HAWKS CL; WANG L; ZHANG C; HORNSBY PJ.: "Progressive loss of malignant behavior in telomerase- negative tumorigenic adrenocortical cells and restoration of tumorigenicity by human telomerase reverse transcriptase", CANCER RES., vol. 64, no. 17, 1 September 2004 (2004-09-01), pages 6144 - 51
TAJIMA K; ITO Y; DEMACHI A; NISHIDA K; AKATSUKA Y; TSUJIMURA K; HIDA T; MORISHIMA Y; KUWANO H; MITSUDOMI T: "Interferon-gamma differentially regulates susceptibility of lung cancer cells to telomerase-specific cytotoxic T lymphocytes", INT J CANCER, vol. 110, no. 3, 20 June 2004 (2004-06-20), pages 403 - 12
TARTOUR ET AL., IMMUNOL LETT, vol. 74, no. 1, 15 September 2000 (2000-09-15), pages 1 - 3
TARTOUR ET AL., PRESSE MED., vol. 25, no. 25, 16 November 1996 (1996-11-16), pages 1717 - 22
VONDERHEIDE ET AL., CLIN CANCER RES, vol. 10, 2004, pages 828 - 39
VONDERHEIDE ET AL., CLIN CANCER RES, vol. 7, 2001, pages 3343 - 8
VONDERHEIDE ET AL., IMMUNITY, vol. 10, 1999, pages 673 - 9
VONDERHEIDE RH.: "Telomerase as a universal tumor-associated antigen for cancer immunotherapy", ONCOGENE, vol. 21, no. 4, 21 January 2002 (2002-01-21), pages 674 - 9
VONDERHEIDE RH; ANDERSON KS; HAHN WC; BUTLER MO; SCHULTZE JL; NADLER LM.: "Characterization of HLA-A3-restricted cytotoxic T lymphocytes reactive against the widely expressed tumor antigen telomerase", CLIN CANCER RES., vol. 7, no. 11, November 2001 (2001-11-01), pages 3343 - 8
VONDERHEIDE RH; DOMCHEK SM; SCHULTZE JL; GEORGE DJ; HOAR KM; CHEN DY; STEPHANS KF; MASUTOMI K; LODA M; XIA Z: "Vaccination of cancer patients against telomerase induces functional antitumor CD8+ T lymphocytes", CLIN CANCER RES., vol. 10, no. 3, 1 February 2004 (2004-02-01), pages 828 - 39
VONDERHEIDE RH; HAHN WC; SCHULTZE JL; NADLER LM: "The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T ymphocytes", MMUNITY, vol. 10, no. 6, June 1999 (1999-06-01), pages 673 - 9
VONDERHEIDE RH; SCHULTZE JL; ANDERSON KS; MAECKER B; BUTLER MO; XIA Z; KURODA MJ; VON BERGWELT-BAILDON MS; BEDOR MM; HOAR KM: "Equivalent induction of telomerase-specific cytotoxic T lymphocytes from tumor-bearing patients and healthy individuals", CANCER RES., vol. 61, no. 23, 1 December 2001 (2001-12-01), pages 8366 - 70
ZENNOU ET AL., CELL, vol. 14, no. 101, 2000, pages 173

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10383928B2 (en) 2010-02-16 2019-08-20 Ultimovacs As Telomerase polypeptide vaccine for treating cancer
US11529403B2 (en) 2010-02-16 2022-12-20 Ultimovacs As Telomerase polypeptide vaccine for treating cancer
US11998595B2 (en) 2010-02-16 2024-06-04 Ultimovacs Asa Telomerase polypeptide vaccine for treating cancer

Also Published As

Publication number Publication date
EP1910529A2 (de) 2008-04-16
EP2626420B1 (de) 2016-06-22
JP2016104031A (ja) 2016-06-09
WO2007014740A2 (en) 2007-02-08
CA2616674A1 (en) 2007-02-08
EP1910529B1 (de) 2013-10-16
JP5931812B2 (ja) 2016-06-08
US8003773B2 (en) 2011-08-23
EP2626420A3 (de) 2013-10-30
JP2009502150A (ja) 2009-01-29
US20100172878A1 (en) 2010-07-08
US20090175892A1 (en) 2009-07-09
US8858931B2 (en) 2014-10-14
JP2013230164A (ja) 2013-11-14
JP2012183076A (ja) 2012-09-27
DK1910529T3 (da) 2014-01-13
PT2626420T (pt) 2016-09-28
JP6257667B2 (ja) 2018-01-10
CA2616674C (en) 2018-09-04
JP5631545B2 (ja) 2014-11-26
WO2007014740A3 (en) 2007-05-03
ES2440768T3 (es) 2014-01-30
DK2626420T3 (en) 2016-09-26
US20140056932A1 (en) 2014-02-27
US9624479B2 (en) 2017-04-18
EP1748067A1 (de) 2007-01-31
JP5836202B2 (ja) 2015-12-24
ES2590462T3 (es) 2016-11-22
PL2626420T3 (pl) 2017-01-31

Similar Documents

Publication Publication Date Title
EP1910529B1 (de) Für hla-b7-restringierte htert-epitope codierende polynukleotide zur anwendung in der prevention und/oder behandlung von krebs
JP5491315B2 (ja) テロメラーゼ由来抗原ペプチド
US6440735B1 (en) Dendritic cell vaccine containing telomerase reverse transcriptase for the treament of cancer
US20120269858A1 (en) Polypeptides
JP2017502649A (ja) テロメラーゼをコードするｄｎａワクチン
US7824849B2 (en) Cellular telomerase vaccine and its use for treating cancer
EP1207199B1 (de) Tumor antigen
US10767167B2 (en) Polynucleotides encoding MHC class I-restricted hTERT epitopes, analogues thereof or polyepitopes
WO2010114129A1 (ja) Ｔ細胞レセプター及び該レセプターをコードする核酸
Li et al. Uses of telomerase peptides in anti-tumor immune therapy

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AC Divisional application: reference to earlier application

Ref document number: 1910529

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

PUAL Search report despatched

Free format text: ORIGINAL CODE: 0009013

AK Designated contracting states

Kind code of ref document: A3

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

RIC1 Information provided on ipc code assigned before grant

Ipc: C12N 9/12 20060101AFI20130926BHEP

17P Request for examination filed

Effective date: 20140430

RBV Designated contracting states (corrected)

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

17Q First examination report despatched

Effective date: 20140925

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20160104

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AC Divisional application: reference to earlier application

Ref document number: 1910529

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 807692

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160715

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 11

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602006049429

Country of ref document: DE

REG Reference to a national code

Ref country code: NL

Ref legal event code: FP

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

Effective date: 20160922

REG Reference to a national code

Ref country code: PT

Ref legal event code: SC4A

Ref document number: 2626420

Country of ref document: PT

Date of ref document: 20160928

Kind code of ref document: T

Free format text: AVAILABILITY OF NATIONAL TRANSLATION

Effective date: 20160921

REG Reference to a national code

Ref country code: CH

Ref legal event code: NV

Representative=s name: BOHEST AG, CH

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160622

REG Reference to a national code

Ref country code: ES

Ref legal event code: FG2A

Ref document number: 2590462

Country of ref document: ES

Kind code of ref document: T3

Effective date: 20161122

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160923

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160622

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20161022

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160622

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160622

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160622

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602006049429

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160622

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

26N No opposition filed

Effective date: 20170323

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 12

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160622

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160622

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20060731

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 13

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20160622

REG Reference to a national code

Ref country code: AT

Ref legal event code: UEP

Ref document number: 807692

Country of ref document: AT

Kind code of ref document: T

Effective date: 20160622

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: PT

Payment date: 20230619

Year of fee payment: 18

Ref country code: NL

Payment date: 20230622

Year of fee payment: 18

Ref country code: IE

Payment date: 20230619

Year of fee payment: 18

Ref country code: DK

Payment date: 20230626

Year of fee payment: 18

Ref country code: CZ

Payment date: 20230621

Year of fee payment: 18

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: PL

Payment date: 20230620

Year of fee payment: 18

Ref country code: LU

Payment date: 20230622

Year of fee payment: 18

Ref country code: FI

Payment date: 20230626

Year of fee payment: 18

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: TR

Payment date: 20230708

Year of fee payment: 18

Ref country code: IT

Payment date: 20230707

Year of fee payment: 18

Ref country code: GB

Payment date: 20230725

Year of fee payment: 18

Ref country code: ES

Payment date: 20230807

Year of fee payment: 18

Ref country code: CH

Payment date: 20230801

Year of fee payment: 18

Ref country code: AT

Payment date: 20230621

Year of fee payment: 18

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: SE

Payment date: 20230721

Year of fee payment: 18

Ref country code: FR

Payment date: 20230727

Year of fee payment: 18

Ref country code: DE

Payment date: 20230712

Year of fee payment: 18

Ref country code: BE

Payment date: 20230721

Year of fee payment: 18